Ethanol alters rodent iNOS activity by changing Nos2 gene expression. No published data exists for ethanol effects on human iNOS activity. We have data from human A172 astrocytoma cells showing a biphasic effect of low and high ethanol concentrations on iNOS activity. The working hypothesis is that the biphasic effect of ethanol on human astrocyte iNOS activity is due to a biphasic effect on NOS2 gene expression. Disruption of NOS2 gene expression by ethanol would result in either increased or decreased production of iNOS and subsequent changes in nitric oxide generation. The CNS immune response would then be compromised resulting in increased susceptibility to infection and brain damage. This project will provide a crucial component of a long-term goal designed to lend insight into the effect of alcohol on CNS genes involved in the pathology of brain disorders. The specific aims are: (1) to further characterize the effects of acute and chronic in vitro ethanol exposure on iNOS activity, protein expression, and mRNA levels in normal human astrocytes (NHA) and human A172 astrocytoma cells; and (2) to determine the mechanism underlying the biphasic acute and chronic effects of ethanol on human NOS2 gene expression. Effects of acute and chronic ethanol exposure on cytokine-induced NOS2 expression will be determined by assessing nitrite production, iNOS protein and mRNA levels. Effects on NOS2 promoter activity will be studied by transfection of A172 cells with luciferase reporter gene constructs. Use of deletion constructs will help identify ethanol-sensitive regions for future studies on trans-acting factors involved in the action of ethanol.